An experimental in silico study on COVID‐19: Response of neutrophil‐related genes to antibiotics

Abstract Background and Aims All components of the immune system are involved in alleviating severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. Further research is required to provide detailed insights into COVID‐19‐related immune compartments and pathways. In addition, a significant percentage of hospitalized COVID‐19 patients suspect bacterial infections and antimicrobial resistance occurs following antibiotics treatment. The aim of this study was to evaluate the possible effects of antibiotics on the response of neutrophil‐related genes in SARS‐CoV‐2 patients by an experimental in silico study. Methods The two data sets GSE1739 and GSE21802 including 10 SARS positive patients and 35 influenza A (H1N1) patients were analyzed, respectively. Differentially expressed genes (DEGs) between these two data sets were determined by GEO2R analysis and the Venn diagram online tool. After determining the hub genes involved in immune responses, the expression of these genes in 30 COVID‐19 patients and 30 healthy individuals was analyzed by real‐time polymerase chain reaction (PCR). All patients received antibiotics, including levofloxacin, colistin, meropenem, and ceftazidime. Results GEO2R analysis detected 240 and 120 DEGs in GSE21802 and GSE1739, respectively. Twenty DEGs were considered as enriched hub genes involved in immune processes such as neutrophil degranulation, neutrophil activation, and antimicrobial humoral response. The central nodes were attributed to the genes of neutrophil elastase (ELANE), arginase 1 (ARG‐1), lipocalin 2 (LCN2), and defensin 4 (DEFA4). Compared to the healthy subjects, the expression of LCN2 and DEFA4 were significantly reduced in COVID‐19 patients. However, no significant differences were observed in the ELANE and AGR‐1 levels between COVID‐19 subjects and the control group. Conclusions Activation and degranulation of neutrophils were observed mainly in SARS, and H1N1 infection processes and antibiotics administration could affect neutrophil activity during viral infection. It can be suggested that antibiotics can decrease inflammation by restoring the expression of neutrophil‐related genes in COVID‐19 patients.

and death. 4,5 Innate and adaptive immune responses are essential for eliminating CoVs infected cells. The primary antiviral defense mechanism is associated with the production of interferon (IFN) Types I and III and various chemokines. These chemokines induce other innate response cells including, leukocytes, monocytes, natural killer (NK) cells, dendritic cells (DCs), and eventually recruit lymphocytes to eventually viral antigens to DCs. 6 Based on the available findings, the inflammatory response and subsequent immunity in the early stages of COVID-19 infection are comparable to other coronaviruses. 7 These findings were further confirmed by increased serum levels of these molecules in COVID-19 patients. Thus, SARS-CoV-2 can escape the antiviral defense system, activate the innate response, and utilize compatible immune cells. In terms of immunopathogenesis, the role of neutrophils in the development of COVID-19 is highlighted. 8 Neutrophils are the most abundant granulocytes involved in the innate immune system and effectively respond to various bacterial and fungal infections. The role of neutrophils in the viral defense process refers to interactions with other immune mechanisms such as cytokine release, virus internalization, and killing mechanisms. 9 However, neutrophilia has been reported as an indicator of severe respiratory symptoms and a poor outcome in COVID-19 patients. Moreover, significant neutrophil infiltration has been identified in COVID-19 patients. 10 Along with the mechanism of infiltration, the pathological effects of neutrophil extracellular traps (NETs) on various inflammatory conditions such as respiratory failure are reported. Indeed, in response to infection, neutrophils release NETs that are composed of extracellular DNA fibers, histones, antimicrobial proteins, proteases such as neutrophil elastase, and oxidant enzymes. Therefore, disruption of the regulation of these mechanisms can cause inflammation. 11 Neutrophil elastase (ELANE), Arginase 1 (ARG-1), Lipocalin 2 (LCN2), and Defensin 4 (DEFA4) are four vital genes involved in neutrophil-mediated immunity. Bioinformatic studies have identified these genes as vital components that are significantly upregulated during SARS infection. 12 Besides this, overexpression of ELANE has been determined in the nasopharyngeal swabs of COVID-19 patients. Likewise, upregulated expression of ARG-1 has been detected in the blood samples and nasopharyngeal aspirates of these patients as well. 13,14 In addition, dysregulated immune response to bacterial infections is an essential issue in COVID-19 patients. Clinical evidence confirms bacterial coinfections in the hospitalized COVID-19 patients who have previously received antibiotics. 15 Excessive intake of antibiotics in patients with SARS-CoV-2 infection, which could lead to antimicrobial resistance has to be considered as well. Ultimately, the elucidation of how ELANE, ARG-1, LCN2, and DEAF4 act as innate immunity mediators along with antibiotic effects in COVID-19 infection can provide effective therapeutic strategies. Venn diagram online tool (http://bioinformatics.psb.ugent.be) to identify common DEGs between the two mentioned data sets.

| Gene ontology and protein-protein interaction (PPI network analysis)
To enrich the gene ontology, the hub genes were subjected to gProfiler database (https://biit.cs.ut.ee/gprofiler/) 17 and the Cytoscape ClueGO tool. 18 Accordingly, the analysis mode was set as functional and the virtual style was considered significant. In addition, hub genes were assessed by Cytoscape String app 19 to predict the probable PPI of the hubs (confidence score ≥ 0.5 and interactors = 0, as the cut-off criteria). Finally, the Cytoscape Network Analyzer tool was used to examine the interaction number of each hub and other network information.

| Sampling
This study was approved by the Ethics Committee of the Medical Genetics Research Center of Genome, Isfahan, Iran. Thirty COVID-19 patients including 16 men and 14 women, with a mean age of 60.9 ± 9.8 years were enrolled from Alzahra Hospital (Isfahan, Iran).
Also, 30 random samples including 13 men and 17 women with a mean age of 58.9 ± 9.4 years participated as healthy subjects for the control group. COVID-19 patients were admitted to the intensive care unit (ICU) with severe symptoms diagnosed by an infectious disease specialist (inclusion criteria). Patients neither had a history of underlying disease nor autoimmune disorders (exclusion criteria). All patients were treated with antibiotics including Levofloxacin, Colistin, Meropenem, and Ceftazidime. Whole blood samples (5 ml) were collected in EDTA-containing ice-cold tubes.

| Isolation of total RNA from human whole blood
Total RNA was extracted using RNA Extraction-Kit (Favor-Prep, Blood/Cultured Cell Total RNA) according to the manufacturer's instructions as follows.
The red blood cells were lysed by adding whole human blood (200-300 μl) preserved with an anticoagulant to a tube. The RL buffer (5:1 ratio) was then added to each sample and was mixed with inversion followed by 10 min of incubation on ice. Samples were centrifuged at 4500 rpm for 1 min to collect cell pellets. The cell pellets were resuspended using an RL buffer followed by a vortex and further centrifuged. The supernatant was completely discarded and then FARB Buffer and β-Mercaptoethanol were added to each sample. The samples were vigorously vortexed. The filter column was placed on a collection tube and the samples were transferred to the filter column centrifuged at 18,000g for 2 min. The clarified supernatants were transferred to a new tube and RNase-free ethanol (70%) was added, mixed well, and transferred to the FARB Mini column. The samples were centrifuged at full speed and flow was discarded. The wash buffer was then added to the FARB mini-column and centrifuged at full speed. RNase-free DNase 1 solution (0.5 U/μl) was added to the center of the FARB mini-column membrane. After 15 min, the washing step was carried out twice using a wash buffer, and samples were centrifuged (full speed, 1 min). The FARB minicolumn was dried to prevent liquid residue. The column was placed in a wash tube and RNase-free ddH 2 O (40-100 µl) was added to the center of the column membrane and centrifuged at full speed for 1 min to wash the RNA. The quality of the extracted RNA was measured at a wavelength ratio of 260.280 nm using a NanoDrop spectrometer (Thermo Scientific).

| Complementary DNA (cDNA) synthesis
cDNA was synthesized using Bio-fact standard kit (Biofact) and the process was conducted on Applied Biosystems ® Veriti ® 96-Well Thermal Cycler. The following mixture was prepared in a polymerase chain reaction (PCR) tube with a total volume of 20 µl consisting of total RNA (~10 ng), random hexamer (50 µM), 2× RT Pre-Mix, RNAase-free water. The mixture was incubated at room temperature for 5 min followed by 30 min of incubation at 50°C to complete cDNA synthesis.

| Quantitative real-time PCR (qPCR)
Expression of LCN2, DEFA4, ELANE, and ARG-1 was assessed by real-time PCR using specific primers (Table 1, Figure S1) and SYBR Green dye (Biofact) conducted on Rotor-Gene 6000 instrument (Corbett Life Science). The qPCR results were analyzed based on the ∆∆ 2 C − t method. Expression of glyceraldehyde-3-phosphate dehydrogenase was also evaluated as a reference gene.

| Statistical analysis
The results were statistically analyzed and normalized by Graph Pad Prism software version 8.0.2 (Graph Pad) and Shapiro-Wilk test, respectively. The unpaired t-test was used to analyze and normalize the expression level between two groups according to the normally distributed genes data. All results are presented as mean ± standard deviation (SD), and p ≤ 0.05 was considered statistically significant.

| Gene Ontology and PPI network analysis
The gProfiler analyzing reports are listed in Table 2. According to the analysis of the ClueGO tool, the hubs were significantly enriched in crucial immune processes including neutrophil degranulation, neutrophil activation, neutrophil-mediated immunity, leukocyte degranulation, and antimicrobial humoral response ( Figure 3).
PPI network analysis identified the interaction/correlation between the hubs (Figure 4). In addition, network analysis with the Cytoscape analyzer tool identified the network with a confidence score of 0.699 and PPI enrichment p ≤ 1.0e−16, stating that the network has significant interactions beyond expectations. Also, central nodes were determined with criteria of interaction numbers of more than 10 nodes (Table 2). Finally, according to the interaction numbers of hubs and altered expression levels, ELANE, ARG1, DEFA4, and LCN2 were selected for further experimental analysis.  Overexpression of LCN2 in respiratory syncytial virus infection has been reported to be a very severe viral infection. 27 DEFA4 is mainly expressed in neutrophils to increase virus uptake. 28 However, these hubs could recruit inflammatory cells and participate in all phases of innate and adaptive immune responses in the lung that include initial deterioration of pathogens, mounting, and resolution. Furthermore, anti-inflammatory effects of the investigated are reported as well.
Recent evidence suggests that secreted α-defensins could prohibit various respiratory viruses, including RSV, adenovirus, and parainfluenza virus. 28 This antiviral effect could serve a novel field in the development of novel therapeutic strategies for viral infections. [29][30][31] ARG1 expression in myeloid cells has emerged as a prominent regulator of innate and adaptive immune responses. In addition to wound healing properties, ARG1 activity can also suppress the antiviral immune response during some viral infections. 32 ELANE is another important gene that is overexpressed in COVID-19 patients.
Neutrophils are known to be part of inflammatory responses that secrete elastin during viral infection. Increased elastase activity during viral infections could result in detrimental outcomes in pulmonary injury associated with the pathogenesis of chronic obstructive pulmonary disease, cystic fibrosis, ARDS, and pulmonary fibrosis. 33,34 Decreasing neutrophil load and increasing host defenses due to ELANE inhibition propose this system to protect the lungs in severe SARS-CoV patients. Accordingly, timely control of cytokine storms in the early stages of virus entry is the basis for improving the treatment of COVID-19 patients. 35

| CONCLUSION
In conclusion, this investigation suggests that deregulated DEGs in viral infections such as SARS and influenza belong to innate immune components, particularly neutrophils. In addition, prescribing antibiotics to SARS-CoV-2 patients may decrease inflammation and pneumonia by restoring neutrophil-related genes. However, to confirm these results and to clarify the treatment and prevention of SARS-CoV-2 infection, further studies in larger population sizes.
In addition, it could be suggested for subsequent experiments to analyze gene expression in pulmonary neutrophils in severe COVID-19 patients and compare with levels in peripheral blood neutrophils.

ACKNOWLEDGMENTS
The authors would like to appreciate the Alzahra University Hospital staff for support of this study. This study was supported by the Medical Genetics Research Center of Genome, Isfahan University of medical sciences, Isfahan, Iran (Grant number 199134).

CONFLICTS OF INTEREST
The authors declare no conflicts of interest.

ETHICS STATEMENT
The informed written consent was obtained from the patient's parent or legal guardian. This study was approved by the Ethics Committee access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.

DATA AVAILABILITY STATEMENT
The authors confirm that the data supporting the findings of this study are available within the article.